This research concerns 4 related topics: (1) A methodological investigation of postlabeling methods for base composition and sequence analysis of nonradioactive RNA. Procedures developed with support by this grant will be further refined, with emphasis on increased sensitivity. (2) Comparative studies on tRNAs from normal and cancerous tissues. Ongoing work which has provided evidence for such differences will be continued. (3) Studies on the actions of 5-azacytidine, 5-fluorocytidine and related analogs on tRNA and DNA. 5-azacytidine was first shown by us to be a specific inhibitor of the synthesis of 5-methylcytosine in nucleic acids. Ongoing studies will be continued, with emphasis on the mechanism of this effect and its functional implications for protein synthesis. (4) Analysis of structural and functional effects of additional drugs which are potentially capable of causing undermethylation of nucleic acids. Compounds will include ethionine, cycloleucine, adenosine dialdehyde, 3-deazaadenosine, 5-fluorocytidine dialdehyde, and 5-fluorouridine dialdehyde. Our major long-term objectives are: (1) to elucidate structural alterations of nucleic acids, especially tRNA, in cancer cells, and possible functional implications of such alterations, (2) to study effects of antimetabolites on nucleic acid structure and functions, and (3) to attempt to define roles of modified nucleotides in mammalian tRNA by comparing deficient tRNAs from cancerous or drug-treated tissues with their normal counterparts. The proposed research, it is hoped, will thus lead to new knowledge concerning nucleic acid alterations induced by drugs and by neoplastic transformation, structure-function relationships of mammalian tRNAs, the roles of modified nucleotides, and the mechanisms of action of anticancer drugs.